Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides

ABSTRACT

The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.

This invention was made with government grant support under GM 22341awarded by National Institutes of Health. The government has certainrights in the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/349,510,filed Dec. 2, 1994, now U.S. Pat. No. 5,723,323 which is a continuationof application Ser. No. 08/133,952, filed Oct. 8, 1993, now abandoned,which is a continuation of application Ser. No. 07/977,307, filed Nov.16, 1992, now abandoned, which is a continuation of application Ser. No.07/616,319, filed Nov. 21, 1990, now abandoned, which is a continuationof application Ser. No. 06/942,630, filed as PCT/CH85/00099 Jun. 17,1985, now abandoned, the entire disclosures all of which areincorporated herein by reference.

The present invention has as its object a process for obtaining DNA,RNA, peptides, polypeptides, or proteins, through use of transformedhost cells containing genes capable of expressing these RNAs, peptides,polypeptides, or proteins; that is to say, by utilization of recombinantDNA technique.

The invention aims in particular at the production of stochastic genesor fragments of stochastic genes in a fashion to permit obtainingsimultaneously, after transcription and translation of these genes, avery large number (on the order of at least 10,000) of completely newproteins, in the presence of host cells (bacterial or eukaryotic)containing these genes respectively capable of expressing theseproteins, and to carry out thereafter a selection or screen among thesaid clones, in order to determine which of them produce proteins withdesired properties, for example, structural, enzymatic, catalytic,antigenic, pharmacologic, or properties of liganding, and moregenerally, chemical, biochemical, biological, etc. properties.

The invention also has as its aim procedures to obtain, sequences of DNAor RNA with utilizable properties notably chemical, biochemical, orbiological properties.

It is clear, therefore, that the invention is open to a very largenumber of applications in very many areas of science, industry, andmedicine.

The process for production of peptides or polypeptides according to theinvention is characterized in that one produces simultaneously, in thesame medium, genes which are at least partially composed of syntheticstochastic polynucleotides, that one introduces the genes thus obtainedinto host cells, that one cultivates simultaneously the independentclones of the transformed host cells containing these genes in such amanner so as to clone the stochastic genes and to obtain the productionof the proteins expressed by each of these stochastic genes, that onecarries out selection and/or screening of the clones of transformed hostcells in a manner to identify those clones producing peptides orpolypeptides having at least one desired activity, that one thereafterisolates the clones thus identified and that one cultivates them toproduce at least one peptide or polypeptide having the said property.

In a first mode of carrying out this process, stochastic genes areproduced by stochastic copolymerization of the four kinds ofdeoxyphosphonucleotides, A, C, G and T from the two ends of an initiallylinearized expression vector, followed by formation of cohesive ends insuch a fashion as to form a stochastic first strand of DNA constitutedby a molecule of expression vector possessing two stochastic sequenceswhose 3' ends are complementary, followed by the synthesis of the secondstrand of the stochastic DNA.

In a second mode of carrying out this process, stochastic genes areproduced by copolymerization of oligonucleotides without cohesive ends,in a manner to form fragments of stochastic DNA, followed by ligation ofthese fragments to a previously linearized expression vector.

The expression vector can be a plasmid, notably a bacterial plasmid.Excellent results have been obtained using the plasmid pUC8 as theexpression vector.

The expression vector can also be viral DNA or a hybrid of plasmid andviral DNA.

The host cells can be prokaryotic cells such as HB 101 and C 600, oreukaryotic cells.

When utilizing the procedure according to the second mode mentionedabove, it is possible to utilize oligonucleotides which for a group ofpalindromic octamers.

Particularly good results are obtained by utilizing the following groupof palindromic octamers;

5' GGAATTCC 3'

5' GGTCGACC 3'

5' CAAGCTTG 3'

5' CCATATGG 3'

5' CATCGATG 3'

It is also possible to use oligonucleotides which form a group ofpalindromic heptamers.

Very good results are obtained utilizing the following group ofpalindromic heptamers:

5' XTCGCGA 3'

5' XCTGCAG 3'

5' RGGTACC 3'

where X=A, G, C, or T, and R=A or T

According to a method to utilize these procedures which is particularlyadvantageous, one isolates and purifies the transforming DNA of theplasmids from a culture of independent clones of the transformed hostcells obtained by following the procedures above, then the purified DNAis cut by at least one restriction enzyme corresponding to specificenzymatic cutting site present in the palindromic octamers or heptamersbut absent from the expression vector which was utilized; this cuttingis followed by inactivation of the restriction enzyme, then onesimultaneously treats the ensemble of linearized stochastic DNAfragments thus obtained with T4 DNA ligase, in such a manner to create anew ensemble of DNA containing new stochastic sequences, this newensemble can therefore contain a number of stochastic genes larger thanthe number of genes in the initial ensemble. One then utilizes this newensemble of transforming DNA to transform the host cells and clone thesegenes, and finally utilizes screening and/or selection and isolates thenew clones of transformed host cells and finally these are cultivated toproduce at least one peptide or polypeptide, for example, a new protein.

The property serving as the criterion for selection of the clones ofhost cells can be the capacity of the peptides or polypeptides, producedby a given clone, to catalyze a given chemical reaction.

For instance, for the production of several peptides and/orpolypeptides, the said property can be the capacity to catalyze asequence of reactions leading from an initial group of chemicalcompounds to at least one target compound.

With the aim of producing an ensemble constituted by a plurality ofpeptides and polypeptides which are reflexively autocatalytic, the saidproperty can be the capacity to catalyze the synthesis of the sameensemble from amino acids and/or oligopeptides in an appropriate milieu.

The said property can also be the capacity to modify selectively thebiological or chemical properties of a given compound, for example, thecapacity to selectively modify the catalytic activity of a polypeptide.

The said property can also be the capacity to stimulate, inhibit, ormodify at least one biological function of at least one biologicallyactive compound, chosen, for example, among the hormones,neurotransmitters, adhesion factors, growth factors, and specificregulators of DNA replication and/or transcription and/or translation ofRNA.

The said property can equally be the capacity of the peptide orpolypeptide to bind to a given ligand.

The invention also has as its object the use of the peptide orpolypeptide obtained by the process specified above, for the detectionand/or the titration of a liquid.

According to a particularly advantageous mode of carrying out theinvention, the criterion for selection of the clones of transformed hostcells is the capacity of these peptides or polypeptides to simulate ormodify the effects of a biologically active molecule, for example, aprotein, and screening and/or selection for clones of transformed hostcells producing at least one peptide or polypeptide having thisproperty, is carried out by preparing antibodies against the activemolecule, then utilizing these antibodies after their purification, toidentify the clones containing this peptide or polypeptide, then bycultivating the clones thus identified, separating and purifying thepeptide or polypeptide produced by these clones, and finally bysubmitting the peptide or polypeptide to an in vitro assay to verifythat it has the capacity to simulate or modify the effects of the saidmolecule.

According to another mode of carrying out the process according to theinvention, the property serving as the criterion of selection is that ofhaving at least one epitope similar to one of the epitopes of a givenantigen.

The invention carries over to obtaining polypeptides by the processspecified above and utilizable as chemotherapeutically activesubstances.

In particular, in the case where the said antigen is EGF, the inventionpermits obtaining polypeptides usable for chemotherapeutic treatment ofepitheliomas.

According to a variant of the procedure, one identifies and isolates theclones of transformed host cells producing peptides or polypeptideshaving the property desired, by affinity chromatography againstantibodies corresponding to a protein expressed by the natural part ofthe DNA hybrid.

For example, in the case where the natural part of the hybrid DNAcontains a gene expressing β-galactosidase, one can advantageouslyidentify and isolates the said clones of transformed host cells byaffinity chromatography against anti-β-galactosidase antibodies. Afterexpression and purification of hybrid peptides or polypeptides, one canseparate and isolate their novel parts.

The invention also applies to a use of the process specified above forthe preparation of a vaccine; the application is characterized by thefact that antibodies against the pathogenic agent are isolated, forexample, antibodies formed after injection of the pathogenic agent inthe body of an animal capable of forming antibodies against this agent,and these antibodies are used to identify the clones producing at leastone protein having at least one epitope similar to one of the epitopesof the pathogenic agent, the transformed host cell corresponding tothese clones are cultured to produce these proteins, this protein isisolated and purified from the clones of cells, then this protein isused for the production of a vaccine against the pathogenic agent.

For example in order to prepare an anti-HVB vaccine, one can extract andpurify at least one capsid protein of the HVB virus, inject this proteininto an animal capable of forming antibodies against this protein havingat least one epitope similar to one of the epitopes of the HVB virus,then cultivate the clones of transformed host cells corresponding tothese clones in a manner to produce this protein, isolate and purify theprotein from culture of these clones of cells and utilize the proteinfor the production of an anti-HVB vaccine.

According to an advantageous mode of carrying out the process accordingto the invention, the host cells consist in bacteria such as Escherichiacoli whose genome contains neither the natural gene expressingβ-galactosidase, nor the EBG gene, that is to say, Z⁻, EBG- E. coli. Thetransformed cells are cultured in the presence of X gal and theindicator IPTG in the medium, and cells positive for β-galactosidasefunctions are detected; thereafter, the transforming DNA is transplantedinto an appropriate clone of host cells for large scale culture toproduce at least one peptide or polypeptide.

The property serving as the criterion for selection of the transformedhost cells can also be the capacity of the polypeptides or proteinsproduced by the culture of these clones to bind to a given compound.

This compound can be in particular chosen advantageously among peptides,polypeptides, and proteins, notably among proteins regulating thetranscription activity of DNA.

On the other hand, the said compound can also be chosen among DNA andRNA sequences.

The invention has also as its object those proteins which are obtainedin the case where the property serving as criterion of selection of theclones of transformed host cells consist in the capacity of theseproteins to bind to regulatory proteins controlling transcriptionactivity of the DNA, or else to DNA and RNA sequences.

The invention has, in addition, as an object, the use of a protein whichis obtained in the first particular case above mentioned, as acis-regulatory sequence controlling replication or transcription of aneighboring DNA sequence.

On the other hand, the aim of the invention also includes utilization ofproteins obtained in the second case mentioned to modify the propertiesof transcription or replication of a sequence of DNA, in a cellcontaining the sequence of DNA, and expressing this protein.

The invention has as its object as well as a process of production ofDNA, characterized by simultaneous production in the same medium, ofgenes at least partially composed of stochastic syntheticpolynucleotides, in that the genes thus obtained are introduced intohost cells to produce an ensemble of transformed host cells, in thatscreening and/or selection on this ensemble is carried out to identifythose host cells containing in their genome stochastic sequences of DNAhaving at least one desired property, and finally, in that the DNA fromthe clones of host cells thus identified is isolated.

The invention further has as its object a procedure to produce RNA,characterized by simultaneous production in the same medium, of genes atleast partially composed of stochastic synthetic polynucleotides, inthat the genes thus obtained are introduced into host cells to producean ensemble of transformed host cells, in that the host cells soproduced are cultivated simultaneously, and screening and/or selectionof this ensemble is carried out in a manner to identify those host cellscontaining stochastic sequences of RNA having at least one desiredproperty, and in that the RNA is isolated from the host cells thusidentified. The said property can be the capacity to bind a givencompound, which might be, for example, a peptide or polypeptide orprotein, or also the capacity to catalyze a given chemical reaction, orthe capacity to be a transfer RNA.

Now the process according to the invention will be described in moredetails, as well as some of its applications, with reference tonon-limitative embodiments.

First, we shall describe particularly useful procedures to carry out thesynthesis of stochastic genes, and the introduction of those genes inbacteria to produce clones of transformed bacteria.

I) Direct synthesis on an expression vector

a) Linearization of the vector

30 micrograms, that is, approximately 10¹³ molecules of the pUC8expression vector are linearized by incubation for 2 hours at 37° C.with 100 units of the Pst1 restriction enzyme in a volume of 300 μl ofthe appropriate standard buffer. The linearized vector is treated withphenol-chloroform then precipitated in ethanol, taken up in volume of 30μl and loaded onto a 0.8% agarose gel in standard TEB buffer. Aftermigration in a field of 3V/cm for three hours, the linearized vector iselectro-eluted, precipitated in ethanol, and taken up in 30 μl of water.

b) Stochastic synthesis using the enzyme Terminal Transferase (TdT)

30 ug of the linearized vector are reacted for 2 hours at 37° C. with 30units of TdT in 300 μl of the appropriate buffer, in the presence of 1mM dGTP, 1 mM dCTP, 0.3 mM dTTP and 1 mM dATP. The lower concentrationof dTTP is chosen in order to reduce the frequence of "stop" codons inthe corresponding messenger RNA. A similar result, although somewhatless favorable, can be obtained by utilizing a lower concentration fordATP than for the other deoxynucleotide triphosphates. The progress ofthe polymerization on the 3' of the Pst1 sites is followed by analysison a gel of aliquots taken during the course of the reaction.

When the reaction attains or passes a mean value of 300 nucleotidesadded per 3' extremity, it is stopped and the free nucleotides areseparated from the polymer by differential precipitation or by passageover a column containing a molecular sieve such as Biogel P60. Afterconcentration by precipitation in ethanol, the polymers are subjected toa further polymerization with TdT, first in the presence of dATP, thenin the presence of dTTP. These last two reactions are separated by afiltration on a gel and are carried out for short intervals (30 secondsto 3 minutes) in order to add sequentially 10-30 A followed by 10-30 Tto the 3' ends of the polymers.

c) Synthesis of the second strand of stochastic DNA

Each molecule of vector possess, at the end of the preceding operation,two stochastic sequences whose 3' ends are complementary. The mixture ofpolymers is therefore incubated in conditions favoring hybridization ofthe complementary extremities (150 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1 mMEDTA at 65° for 10 minutes, followed by lowering the temperature to 22°C. at a rate of 3 to 4° C. per hour). The hybridized polymers are thenreacted with 60 units of the large fragment (Klenow) of polymerase 1, inthe presence of the four nucleotide triphosphates (200 mM) at 4° C. fortwo hours. This step accomplishes the synthesis of the second strandfrom the 3' ends of the hybrid polymers. The molecules which result fromthis direct synthesis starting from linearized vector are thereafterutilized transform competent cells.

d) Transformation of competent clones

100 to 200 ml of competent HB 101 of C 600 at a concentration of 10¹⁰cells/ml, are incubated with the stochastic DNA preparation (from above)in the presence of 6 mM CaCl₂, 6 mM Tris-HCl pHG, 6 mM MgCl₂ for 30minutes at 0° C. A temperature shock of 3 minutes at 37° C. is imposedon the mixture, followed by the addition of 400 to 800 ml of NZY culturemedium, without antibiotics. The transformed culture is incubated at 37°C. for 60 minutes, then diluted to 10 litres by addition of NZY mediumcontaining 40 μg/ml of ampicillin. After 3-5 hours of incubation at 37°C., the amplified culture is centrifuged, and the pellet of transformedcells is lyophilysed and stored at -70° C. Such a culture contains 3×10⁷to 10⁸ independent transformants, each containing a unique stochasticgene inserted into the expression vector.

II) Synthetic of stochastic genes starting from oligonucleotides withoutcohesive ends

This procedure is based on the fact that polymerization of judiciouslychosen palindromic oligonucleotides permits construction of stochasticgenes which have no "stop" codon in any of the six possible readingframes, while at the same time assuring a balanced representation oftriplets specifying all amino acids. Further, and to avoid a repetitionof sequence motifs in the proteins which result, the oligonucleotidescan contain a number of bases which is not a multiple of three. Theexample which follows describes the use of one of the possiblecombinations which fulfil these criteria:

a) Choice of a group of octamers

The group of oligonucleotides following:

5' GGAATTCC 3'

5' GGTCGACC 3'

5' CAAGCTTG 3'

5' CCATATGG 3'

5' CATCGATG 3'

is composed of 5 palindromes (thus self-complementary sequences) whereit is easy to verify that their stochastic polymerization does notgenerate any "stop" codons, and specifies all the amino acids.

Obviously, one can utilize other groups of palindromic octamers which donot generate any "stop" codons and specify all the amino acids found inpolypeptides. Clearly, it is also possible to utilize non-palindromicgroups of octamers, or other oligomers, under the condition that theircomplements forming double stranded DNA are also used.

b) Assembly of a stochastic gene from a group of octamers.

A mixture containing 5 μg each of the oligonucleotides indicated above(previously phosphorylated at the 5' position by a standard procedure)is reacted in a 100 μl volume containing 1 mM ATP, 10%polyethyleneglycol, and 100 units of T4 DNA ligase in the appropriatebuffer at 13° C. for six hours. This step carries out the stochasticpolymerization of the oligomers in the double stranded stare and withoutcohesive ends. The resulting polymers are isolated by passage over amolecular sieve (Biogel P60) recovering those with 20 to 100 oligomers.After concentration, this fraction is again submitted to catalysis orpolymerization by T4 DNA ligase under the conditions described above.Thereafter, as described above, those polymers which have assembled atleast 100 oligomers are isolated.

c) Preparation of the host plasmid

The pUC8 expression vector is linearized by Sma1 enzyme in theappropriate buffer, as described above. The vector linearized by Sma1does not have cohesive ends. Thus the linearized vector is treated bycalf intestine alkaline phosphatase (CIP) at a level of one unit permicrogram of vector in the appropriate buffer, at 37° C. for 30 minutes.The CIP enzyme is thereafter inactivated by two successive extractionswith phenol-chloroform. The linearized and dephosphorylated vector isprecipitated in ethanol, then redissolved in water at 1 mg/ml.

d) Ligation of stochastic genes to the vector

Equimolar quantities of vector and polymers are mixed and incubated inthe presence of 1000 units of T4 DNA ligase, 1 mM ATP, 10% polyethyleneglycol, in the appropriate buffer, for 12 hours at 13° C. This stepligates the stochastic polymers in the expression vector and formsdouble stranded circular molecules which are, therefore, capable oftransforming.

Transformation of competent clones

Transformation of competent clones in carried out in the mannerpreviously described.

III) Assembly of stochastic genes starting from a group of heptamers

This procedure differs from that just discussed in that it utilizespalindromic heptamers which have variable cohesive ends, in place ofstochastic sequences containing a smaller number of identical motifs.

a) Choice of a group of heptamers

It is possible, as an example, to use the following three palindromicheptamers:

5' XTCGCGA 3'

5' XCTGCAG 3'

5" RGGTACC 3'

where X=A, G, C, or T and R=A or T, and where polymerization cannotgenerate any "stop" codons and forms triplets specifying all the aminoacids. Clearly, it is possible to use other groups of heptamersfulfilling these same conditions.

b) Polymerization of a group of heptamers

This polymerization is carried out exactly in the fashion describedabove for octamers.

c) Elimination of cohesive extremities

The polymers thus obtained have one unpaired base on their two 5'extremities. Thus, it is necessary to add the complementary base to thecorresponding 3' extremities. This is carried out as follows: 10micrograms of the double stranded polymers are reacted with 10 units ofthe Klenow enzyme, in the presence of the four deoxynucleotidephosphates(200 mM) in a volume of 100 μl, at 4° C., for 60 minutes. The enzyme isinactivated by phenol chloroform extraction, and the polymers arecleansed of the residual free nucleotides by differential precipitation.The polymers are then ligated to the host plasmid (previously linearizedand dephosphorylated) by following the procedures described above.

It is to be noted that the two last procedures which were describedutilize palindromic octamers or heptamers which constitute specificsites of certain restriction enzymes. These sites are absent, for themost part, from the pUC8 expression vector. Thus, it is possible toaugment considerably the complexity of an initial preparation ofstochastic genes by proceeding in the following way: the plasmid DNAderived from the culture of 10⁷ independent transformants obtained byone of the two last procedures described above, is isolated. After thisDNA is purified, it is partially digested by Cla1 restriction enzyme(procedure II) or by the Pst1 restriction enzyme (procedure III). Afterinactivation of the enzyme, partially digested DNA is treated with T4DNA ligase, which has the effect of creating a very large number of newsequences, while conserving the fundamental properties of the initialsequences. This new ensemble of stochastic sequences can then be used totransform competent cells. In addition, the stochastic genes cloned byprocedure II and III can be excised intact from the pUC8 expressionvector by utilizing restriction sites belonging to the cloning vectorand not represented in the stochastic DNA sequences.

Recombination within the stochastic genes generated by the twoprocedures just described, which result from the internal homology dueto the recurrent molecular motifs, is an important additional method toachieve in vivo mutagenesis of the coding sequences. This results in anaugmentation of the number of the new genes which can be examined.

Finally, for all the procedures to generate novel synthetic genes, it ispossible to use a number of common techniques to modify genes in vivo orin vitro, such as a change of reading frame, inversion of sequences withrespect to their promotor, point mutations, or utilization of host cellsexpressing one or several suppressor tRNAs.

In considering the above description, it is clear that it is possible toconstruct, in vitro, an extremely large number (for example, greaterthan a billion) different genes, by enzymatic polymerization ofnucleotides or of oligonucleotides. This polymerization is carried outin a stochastic manner, as determined by the respective concentrationsof the nucleotides or oligonucleotides present in the reaction mixture.

As indicated above, two methods can be utilized to clone such genes (orcoding sequences): the polymerization can be carried out directly on acloning expression vector, which was previously linearized; or it ispossible to proceed sequentially to the polymerization then the ligationof the polymers to the expression vector.

In the two cases, the next step is transformation or transfection ofcompetent bacterial cells (or cells in culture). This step constitutescloning the stochastic genes in living cells where they are indefinitelypropagated and expressed.

Clearly, in addition to the procedures which were described above, it isfeasible to use all other methods which are appropriate for thesynthesis of stochastic sequences. In particular, it is possible tocarry out polymerization, by biochemical means, of single strandedoligomers of DNA or RNA obtained by chemical synthesis, then treat thesesegments of DNA or RNA by established procedures to generate doublestranded DNA (cDNA) in order to clone such genes.

Screening or selection of clones of transformed host cells

The further step of the procedure according to the invention consists inexamining the transformed or transfected cells by selection orscreening, in order to isolate one or several cells whose transformingor transfecting DNA leads to the synthesis of a transcription product(RNA) or translation product (protein) having desired property. Theseproperties can be, for example, enzymatic, functional, or structural.

One of the most important aspects of the process, according to theinvention, is that it permits the simultaneous screening or selection ofan exploitable product (RNA or protein) and the gene which produces thatproduct. In addition, the DNA synthesized and cloned as described, canbe selected or screened in order to isolate sequences of DNAconstituting products in themselves, having exploitable biochemicalproperties.

We shall now describe, as non-limiting examples, preferred proceduresfor screening and/or selection of clones of transformed cells such thatthe novel proteins are of interest from the point of view of industrialor medical applications.

One of these procedures rests in the idea of producing, or obtaining,polyclonal or monoclonal antibodies, by established techniques, directedagainst a protein or another type of molecule of biochemical or medicalinterest, where that molecule is, or has been rendered, immunogenic, andthereafter using these antibodies as probes to identify among the verylarge number of clones transformed by stochastic genes, those whoseprotein react with these antibodies. This reaction is a result of astructural homology which exists between the polypeptide synthesized bythe stochastic gene and the initial molecule. It is possible in this wayto isolate numbers of novel proteins which behave as epitopes orantigenic determinants on the initial molecule. Such novel proteins areliable to simulate, stimulate, modulate, or block the effect of theinitial molecule. It will be clear that this means of selection orscreening may itself have very many pharmacologic and biochemicalapplications. Below we describe, as a non-limiting example, this firstmode of operation in a concrete case:

EGF (epidermal growth factor) is a small protein present in the blood,whose role is to stimulate the growth of epithelial cells. This effectis obtained by the interaction of EGF with a specific receptor situatedin the membrane of epithelial cells. Antibodies directed against EGF areprepared by injecting animals with EGF coupled to KLH (keyhole limpethemocyanin) to augment the immunogenicity of the EGF. The anti-EGFantibodies of the immunized animals are purified, for example, bypassage over an affinity column, where the ligand is EGF or a syntheticpeptide corresponding to a fragment of EGF. The purified anti-EGFantibodies are then used as probes to screen a large number of bacterialclones lysed by chloroform, and on a solid support. The anti-EGFantibodies bind those stochastic peptides or proteins whose epitopesresemble those of the initial antigen. The clones containing suchpeptides or proteins are shown by autoradiography after incubation ofthe solid support with radioactive protein A, or after incubation with aradioactive antibody.

These steps identify those clones, each of which contains one protein(and its gene) reacting with the screening antibody. It is feasible toscreen among a very large number of colonies of bacterial cells or viralplaques (for example, on the order of 1,000,000) and it is feasible todetect extremely small quantities, on the order of 1 nanogram, ofprotein product. Thereafter, the identified clones are cultured and theproteins so detected are purified in conventional ways. These proteinsare tested in vitro in cultures of epithelial cells to determine if theyinhibit, simulate, or modulate the effects of EGF on these cultures.Among the proteins so obtained, some may be utilized for thechemotherapeutic treatment of epitheliomas. The activities of theproteins thus obtained can be improved by mutation of the DNA coding forthe proteins, in ways analogous to those described above. A variant ofthis procedure consists in purifying these stochastic peptides,polypeptides, or proteins, which can be used as vaccines or moregenerally, to confer an immunity against a pathogenic agent or toexercise other effects on the immunological system, for example, tocreate a tolerance or diminish hypersensitivity with respect to a givenantigen, in particular, due to binding of these peptides, polypeptides,or proteins with the antibodies directed against this antigen. It isclear that it is possible to use such peptides, polypeptides, orproteins in vitro as well as in vivo.

More precisely, in the ensemble of novel proteins which react with theantibodies against a given antigen X, each has at least one epitope incommon with X, thus the ensemble has an ensemble of epitopes in commonwith X. This permits utilization of the ensemble or sub-ensemble as avaccine to confer immunity against X. It is, for example, easy to purifyone or several of the capsid proteins of the hepatitis B virus. Theseproteins can then be injected into an animal, for example, a rabbit, andthe antibodies corresponding to the initial antigen can be recovered byaffinity column purification. These antibodies may be used, as describedabove, to identify clones producing at least one protein having anepitope resembling at least one of the epitopes of the initial antigen.After purification, these proteins are used as antigens (either alone orin combination) with the aim of conferring protection against hepatitisB. The final production of the vaccine does not require further accessto the initial pathogenic agent.

Note that, during the description of the procedures above, a number ofmeans to achieve selection or screening have been described. All theseprocedures may require the purification of a particular protein from atransformed clone. These protein purifications can be carried out byestablished procedures and utilize, in particular, the techniques of gelchromatography, by ion exchange, and by affinity chromatography. Inaddition, the proteins generated by the stochastic genes can be clonedin the form of hybrid proteins having, for example, a sequence of theβ-galactosidase enzyme which permits affinity chromatography againstanti-β-galactosidase antibodies, and allows the subsequent cleavage ofthe hybrid part; that is to say, allowing separation of the novel partand the bacterial part of the hybrid protein. Below we describe theprinciples and procedures for selection of peptides or polypeptides andthe corresponding genes, according to a second method of screening orselection based on the detection of the capacity of these peptides orpolypeptides to catalyse a specific reaction.

As a concrete and non-limiting example, screening or selection in theparticular case of proteins capable of catalyzing the cleavage oflactose, normally a function fulfilled by enzyme β-galactosidase (β-gal)will be described.

As above described, the first step of the process consists in generatinga very large ensemble of expression vectors, each expressing a distinctnovel protein. To be concrete, for example, one may choose the pUC8expression vector with cloning of stochastic sequences of DNA in thePst1 restriction site. The plasmids thus obtained are then introducedinto a clone of E. coli from whose genome the natural gene forβ-galactosidase, Z, and a second gene EBG, unrelated to the first butable to mutate towards β-gal function, have both been eliminated byknown genetic methods. Such host cells (Z⁻, EBG⁻) are not able bythemselves to catalyse lactose hydrolysis, and as a consequence, to uselactose as a carbon source for growth. This permits utilization of suchhost clones for screening or selection for β-gal function.

A convenient biological assay to analyze transformed E. coli clones forthose which have novel genes expressing a β-gal function consists in theculture of bacteria transformed as described in petri dishes containingX-gal in the medium. In this case, all bacterial colonies expressing aβ-gal function are visualized as blue colonies. By using such abiological assay, it is possible to detect even weak catalytic activity.The specific activity of characteristic enzymes ranges from 10 to 10,000product molecules per second.

Supposing that a protein synthesized by a stochastic gene has a weakspecific activity, on the order of one molecule per 100 seconds, itremains possible to detect such catalytic activity. In a petri dishcontaining X-gal in the medium, and the presence of thenon-metabolizable inducer 1PTG (isopropyl-D-thiogalactoside)visualization of a blue region requires cleavage of about 10¹⁰ to 10¹¹molecules of X-gal per square millimeter. A bacterial colony expressinga weak enzyme and occupying a surface area of 1 mm² has about 10⁷ to 10⁸cells. If each cell has only one copy of the weak enzyme, each cellwould need to catalyse cleavage of between 10,000 and 100 of X-gal to bedetected, which would require between 2.7 and 270 hours. Since underselective conditions it is possible to amplify the number of copies ofeach plasmid per cell from 5 to 20 copies per cell, or even to 100 or1000, and because up to 10% of the protein of the cell can be specifiedby the new gene, the duration needed to detect a blue colony in the caseof 100 enzyme molecules of weak activity per cell is on the order of0.27 to 2.7 hours.

As a consequence of these facts, screening a very large number ofindependent bacterial colonies, each expressing a different novel gene,and using the capacity to express a β-gal function as the selectioncriterion, is fully feasible. It is possible to carry out screening ofabout 2000 colonies in one Petri dish of 10 cm diameter. Thus, about 20million colonies can be screened on a sheet of X-gal agar of 1 squaremeter.

It is to be noted that bacterial colonies which appear blue on X-galPetri dishes might be false positives due to a mutation in the bacterialgenome which confers upon it the capacity to metabolize lactose, or forother reasons than those which result from a catalytic activity of thenovel protein expressed by the cells of the colony. Such false positivescan be directly eliminated by purifying the DNA of the expression vectorfrom the positive colony, and retransforming Z⁻, EBG⁻ E. coli hostcells. If the β-gal activity is due to the novel protein coded by thenew gene in the expression vector, all those cells transformed by thatvector will exhibit β-gal function. In contrast, if the initial bluecolony is due to a mutation in the genome of the host cell, it is a rareevent and independent of the transformation, thus the number of cells ofthe new clone of the transformed E. coli capable of expressing galfunction will be small or zero.

The power of mass simultaneous purification of all the expressionvectors from all the positive clones (blue) followed by retransformationof naive bacteria should be stressed. Suppose that the aim is to carryout a screening to select proteins having a catalytic function, and thatthe probability that a new peptide or polypeptide carries out thisfunction at least weakly is 10⁻⁶, while the probability that a clone ofthe E. coli bacterial host undergoes a mutation rendering it capable ofcarrying out the same function is 10⁻⁵, then it can be calculated thatamong 20 million transformed bacteria which are screened, 20 positiveclones will be attributable to the novel genes in expression vectorswhich each carries, while 200 positive clones will be the result ofgenomic mutation. Mass purification of the expression vectors from thetotal of 220 positive bacterial clones followed by retransformation ofthe naive bacteria with the mixture of these expression vectors willproduce a large number of positive clones consisting of all thosebacteria transformed with the 20 expression vectors which code for thenovel proteins having the desired function, and a very small number ofbacterial clones resulting from genomic mutations and containing the 200expression vectors which are not of interest. A small number of cyclesof purification of expression vectors from positive bacterial colonies,followed by such retransformation, allows the detection of very rareexpression vectors truly positive for a desired catalytic activity,despite a high background rate of mutations in the host cells for thesame function.

Following screening operations of this type, it is possible to purifythe new protein by established techniques. The production of thatprotein in large quantity is made possible by the fact thatidentification of the useful protein occurs together with simultaneousidentification of the gene coding for the same protein. Consequently,either the same expression vector can be used, or the novel gene can betransplanted into a more appropriated expression vector for itssynthesis and isolation in large quantity.

It is feasible to apply this method of screening for any enzymaticfunction for which an appropriate biological assay exists. For suchscreenings, it is not necessary that the enzymatic function which issought be useful to the host cell. It is possible to carry outscreenings not only for an enzymatic function but for any other desiredproperty for which it is possible to establish an appropriate biologicalassay. It is thus feasible to carry out, even in the simple case ofβ-gal function visualized on an X-gal Petri plate, a screening of on theorder of 100 million, or even a billion novel genes for a catalyticactivity or any other desired property.

Selection of transformed host cells

On the other hand, it is possible to use selection techniques for anyproperty, catalytic or otherwise, where the presence or absence of theproperty can be rendered essential for the survival of the host cellscontaining the expression vectors which code for the novel genes, oralso can be used to select for those viruses coding and expressing thedesired novel gene. As a non-limiting, but concrete example, selectionfor β-galactosidase function shall be described. An appropriate clone ofZ⁻ EBG⁻ E. coli is not able to grow on lactose as the sole carbonsource. Thus, after carrying out the first step described above, it ispossible to culture a very large number of host cells transformed by theexpression vectors coding for the novel genes, under selectiveconditions, either by progressive diminution of other sources of carbon,or utilization of lactose alone from the start. During the course ofsuch selection, in vivo mutagenesis by recombination, or by explicitlyrecovering the expression vectors and mutagenizing their novel genes invitro by various mutagens, or by any other common technique, permitsadaptive improvements in the capacity to fulfill the desired catalyticfunction. When both selection techniques and convenient bioassaytechniques exist at the same time, as in the present case, it ispossible to use selection techniques initially to enrich therepresentation of host bacteria expressing the β-gal function, thencarry out a screening on a Petri plate on X-gal medium to establishefficiently which are the positive cells. In the absence of convenientbioassays, application of progressively stricter selection is theeasiest route to purify one or a small number of distinct host cellswhose expression vectors code for the proteins catalyzing the desiredreaction.

It is possible to utilize these techniques to find novel proteins havinga large variety of structural and functional characteristics beyond thecapacity to catalyse a specific reaction. For example, it is possible tocarry out a screen or select for novel proteins which bind tocis-regulatory sites on the DNA and thereby block the expression of oneof the host cell's functions, or block transcription of the DNA,stimulate transcription, etc.

For example, in the case of E. coli, a clone mutant in the repressor ofthe lactose operon (i-) expresses β-gal function constitutively due tothe fact the lactose operator is not repressed. All cells of this typeproduce blue clones on Petri plates containing X-gal medium. It ispossible to transform such host cells with expression vectorssynthesizing novel proteins and carry out a screen on X-gal Petri platesin order to detect those clones which are not blue. Among those, somerepresent the case where the new protein binds to the lactose operatorand represses the synthesis of β-gal. It is then feasible to massisolate such plasmids, retransform, isolate those clones which do notproduce β-gal, and thereafter carry out a detailed verification.

As mentioned above, the process can be utilized in order to create, thenisolate, not only exploitable proteins, but also RNA and DNA as productsin themselves, having exploitable properties. This results from the factthat, on one hand, the procedure consists in creating stochasticsequences of DNA which may interact directly with other cellular orbiochemical constituents, and on the other hand, these sequences clonedin expression vectors are transcribed into RNA which are themselvescapable of multiple biochemical interactions.

An example of the use of the procedure to create and select for a DNAwhich is useful in itself

This example illustrates selection for a useful DNA, and thepurification and study of the mechanism of action of regulatory proteinswhich bind to the DNA. Consider a preparation of the oestradiolreceptor, a protein obtained by standard techniques. In the presence ofoestradiol, a steroid sexual hormone, the receptor changes conformationand binds tightly to certain specific sequences in the genomic DNA, thusaffecting the transcription of genes implicated in sexualdifferentiation and the control of fertility. By incubating a mixturecontaining oestradiol, its receptor, and a large number of differentstochastic DNA sequences inserted in their vectors, followed byfiltration of the mixture across a nitrocellulose membrane, one has adirect selection for those stochastic DNA sequences binding to theoestradiol-receptor complex, where only those DNAs bound to a proteinare retained by the membrane. After washing and elution, the DNAliberated from the membrane is utilized as such to transform bacteria.After culture of the transformed bacteria, the vectors which theycontain are again purified and one or several cycles of incubation,filtration, and transformation are carried out as described above. Theseprocedures allow the isolation of stochastic sequences of DNA having anelevated affinity for the oestradiol-receptor complex. Such sequencesare open to numerous diagnostic and pharmacologic applications, inparticular, for developing synthetic oestrogens for the control offertility and treatment of sterility.

Creation and selection of an RNA useful in itself

Let there be a large number of stochastic DNA sequences, produced as hasbeen described and cloned in an expression vector. It follows that theRNA transcribed from these sequences in the transformed host cells canbe useful products themselves. As a non-limiting example, it is possibleto select a stochastic gene coding for a suppressor transfer RNA (tRNA)by the following procedure: a large number (≧10⁸) of stochasticsequences are transformed into competent bacterial hosts carrying a"nonsense" mutation in the arg E. gene. These transformed bacteria areplated on minimal medium without arginine and with the selectiveantibiotic for that plasmid (ampicillin if the vector is pUC8). Onlythose transformed bacteria which have become capable of synthesizingarginine will be able to grow. This phenotype can result either from aback mutation of the host genome, or the presence in the cell of asuppressor. It is easy to test each transformed colony to determine ifthe arg+ phenotype is or is not due to the presence of the stochasticgene in its vector; it suffices to purify the plasmid from this colonyand verify that it confers an arg+ phenotype on all arg E. cellstransformed by it.

Selection of proteins capable of catalyzing a sequence of reactions

Below we describe another means of selection, open to independentapplications, based on the principle of simultaneous and parallelselection of a certain number of novel proteins capable of catalyzing aconnected sequence of reactions.

The basic idea of this method is the following: given an initialensemble of chemical compounds considered as building blocks or elementsof construction from which it is hoped to synthesize one or severaldesired chemical compounds by means of a catalyzed sequence of chemicalreactions, there exists a very large number of reaction routes which canbe partially or completely substituted for one another, which are allthermodynamically possible, and which lead from the set of buildingblocks to the desired target compound(s). Efficient synthesis of atarget compound is favored if each step of at least one reaction pathwayleading from the building block compounds to the target compound iscomprised of reactions each of which is catalyzed. On the other hand, itis relatively less important to determine which among the manyindependent or partially independent reaction pathways is catalyzed. Inthe previous description, we have shown how it is possible to obtain avery large number of host cells each of which expresses a distinct novelprotein.

Each of these novel proteins is a candidate to catalyse one or anotherof the possible reactions, in the set of all the possible reactionsleading from the ensemble of building blocks to the target compound. Ifa sufficiently large number of stochastic proteins is present in areaction mixture containing the building block compounds, such that asufficiently large number of the possible reactions are catalyzed, thereis a high probability that one connected sequence of reactions leadingfrom the set of building block compounds to the target compound will becatalyzed by a subset of the novel proteins. It is clear that thisprocedure can be extended to the catalysis not only of one, but ofseveral target compounds simultaneously.

Based on this principle it is possible to proceed as follows in order toselect in parallel a set of novel proteins catalyzing a desired sequenceof chemical reactions:

1. Specify the desired set of compounds constituting the buildingblocks, utilizing preferentially a reasonably large number of distinctchemical species in order to increase the number of potential concurrentreactions leading to the desired target compound.

2. Using an appropriate volume of reaction medium, add a very largenumber of novel stochastic proteins isolated from transformed ortransfected cells synthesizing these proteins. Carry out an assay todetermine if the target compound is formed. If it is, confirm that thisformation requires the presence of the mixture of novel proteins. If so,then the mixture should contain a subset of proteins catalyzing one orseveral reaction pathways leading from the building block set to thetarget compound. Purify and divide the initial ensemble of clones whichsynthesize the set of novel stochastic proteins, the subset which isrequired to catalyse the sequence of reactions leading to the targetcompound.

More precisely, as a non-limiting example, below we describe selectionof novel proteins capable of catalyzing the synthesis of a specificsmall peptide, in particular, a pentapeptide, starting from a buildingblock set constituted of smaller peptides and amino acids. All peptidesare constituted by a linear sequence of 20 different types of aminoacids, oriented from the amino to the carboxy terminus. Any peptide canbe formed in a single step by the terminal condensation of two smallerpeptides (or of two amino acids), or by hydrolysis of a larger peptide.A peptide with M residues can thus be formed by M-1 condensationreactions. The number of reactions, R, by which a set of peptides havinglength 1, 2, 3, . . .M residues can be interconverted is larger than thenumber of possible molecular species, T. This can be expressed asR/T≅M-2. Thus, starting from a given ensemble of peptides, a very largenumber of independent or partially independent reaction pathways leadsto the synthesis of a specific target peptide. Choose a pentapeptidewhose presence can be determined conveniently by some common assaytechnique for example HPLC (liquid phase high pressure chromatography),paper chromatography, etc. Formation of a peptide bond requires energyin a dilute aqueous medium, but if the peptides participating in thecondensation reactions are adequately concentrated, formation of peptidebonds is thermodynamically favored over hydrolysis and occursefficiently in the presence of an appropriate enzymatic catalyst, forexample pepsin or trypsin, without requiring the presence of ATP orother high energy compounds. Such a reaction mixture of small peptideswhose amino acids are marked radioactively to act as tracers with ³ H,¹⁴ C, ³⁵ S, constituting the building block set can be used atsufficiently high concentrations to lead to condensation reactions.

For example, it is feasible to proceed as follows: 15 mg of each aminoacid and small peptides having 2 to 4 amino acids, chosen to constitutethe building block set, are dissolved in a volume of 0.25 ml to 1.0 mlof a 0.1M pH 7.6 phosphate buffer. A large number of novel proteins,generated and isolated as described above are purified from theirbacterial or other host cells. The mixture of these novel proteins isdissolved to a final concentration on the order of 0.8 to 1.0 mg/ml inthe same buffer. 0.25 ml to 0.5 ml of the protein mixture is added tothe mixture of building blocks. This is incubated at 25° C. to 40° C.for 1 to 40 hours. Aliquots of 8 μl are removed at regular intervals,the first is used as a "blank" and taken before addition of the mixtureof novel proteins. These aliquots are analyzed by chromatography usingn-butanol-acetic acid-pyridine-water (930:6:20:24 by volume) as thesolvent. The chromatogram is dried and analyzed by ninhydrin orautoradiography (with or without intensifying screens). Because thecompound constituting the building block set are radioactively marked,the target compound will be radioactive and it will have specificactivity high enough to permit detection at the level of 1-10 ng. Inplace of standard chromatographic analysis, it is possible to use HPLC(high pressure liquid chromatography) which is faster and simpler tocarry out. More generally, all the usual analytic procedures can beemployed. Consequently it is possible to detect a yield of the targetcompound of less than one part per million by weight compared to thecompounds used as initial building blocks.

If the pentapeptide is formed in the conditions described above, but notwhen an extract is utilized which is derived from host cells transformedby an expression vector containing no stochastic genes, the formation ofthe pentapeptide is not the result of bacteria contaminants and thusrequires the presence of a subset of the novel protein in the reactionmixture.

The following step consists in the separation of the particular subsetof cells which contain expression vectors with the novel proteinscatalyzing the sequence of reactions leading to the target pentapeptide.As an example, if the number of reactions forming this sequence is 5,there are about 5 novel proteins which catalyse the necessary reactions.If the clone bank of bacteria containing the expression vectors whichcode for the novel genes has a number of distinct novel genes which ison the order of 1,000,000 all these expression vectors are isolated enmasse and retransformed into 100 distinct sets of 10⁸ bacteria at aratio of vectors to bacteria which is sufficiently low that, on average,the number of bacteria in each set which are transformed is about halfthe number of initial genes, i.e. about 500,000. Thus, the probabilitythat any given one of the 100 sets of bacteria contains the entire setof 5 critical novel proteins is (1/2)⁵ =1/32. Among the 100 initial setsof bacteria, about 3 will contain the 5 critical transformants. In eachof these sets, the total number of new genes is only 500,000 rather than1,000,000. By successive repetitions, the total number of which is about20 in the present case, this procedure isolates the 5 critical novelgenes. Following this, mutagenesis and selection on this set of 5stochastic genes allows improvement of the necessary catalyticfunctions. In a case where it is necessary to catalyse a sequence of 20reactions and 20 genes coding novel proteins need to be isolated inparallel, it suffices to adjust the multiplicity of transformation suchthat each set of 10⁸ bacteria receives 80% of the 10⁶ stochastic genes,and to use 200 such sets of bacteria. The probability that all 20 novelproteins are found in one set is 0.8²⁰ ≅0.015. Thus, about 2 among the200 sets will have the 20 novel genes which are needed to catalyse theformation of the target compound. The number of cycles required forisolation of the 20 novel genes is on the order of 30.

The principles and procedures described above generalize from the caseof peptides to numerous areas of chemistry in which chemical reactionstake place in aqueous medium, in temperature, pH, and concentrationconditions which permit general enzymatic function. In each case it isnecessary to make use of an assay method to detect the formation of thedesired target compound(s). It is also necessary to choose asufficiently large number of building block compounds to augment thenumber of reaction sequences which lead to the target compound.

The concrete example which was given for the synthesis of a targetpentapeptide can also be generalized as follows:

The procedure as described, generates among other products, stochasticpeptides and proteins. These peptides or proteins can act, catalyticallyor in other ways, on other compounds. They can equally constitute thesubstrates on which they act. Thus, it is possible to select (or screen)for the capacity of such stochastic peptides or proteins to interactamong themselves and thereby modify the conformation, the structure orthe function of some among them. Similarly, it is possible to select (orscreen) for the capacity of these peptides and proteins to catalyseamong themselves, hydrolysis, condensation, transpeptidation or otherreactions modifying the peptides. For example, the hydrolysis of a givenstochastic peptide but at least one member of the set of stochasticpeptides and proteins can be followed and measured by radioactivemarking of the given protein followed by an incubation with a mixture ofthe stochastic proteins in the presence of ions such as Mg, Ca, Zn, Feand ATP or GTP. The appearance of radioactive fragments of the markedprotein is then measured as described. The stochastic protein(s) whichcatalyse this reaction can again be isolated, along with the gene(s)producing them, by sequential diminution of the library of transformedclones, as described above.

An extension of the procedure consists in the selection of an ensembleof stochastic peptides and polypeptides capable of catalyzing a set ofreactions leading from the initial building blocks (amino acids andsmall peptides) to some of the peptides or polypeptides of the set. Itis therefore also possible to select an ensemble capable of catalyzingits own synthesis; such a reflexively autocatalytic set can beestablished in a chemostat where the products of the reactions areconstantly diluted, but where the concentration of the building blocksis maintained constant. Alternatively, synthesis of such a set is aidedby enclosing the complex set of peptides in liposomes by standardtechniques. In a hypertonic aqueous environment surrounding suchlipsomes, condensation reactions forming larger peptides lowers theosmotic pressure inside the lipsomes, drives water molecules produced bythe condensation reactions out of the lipsomes, hence favoring synthesisof larger polymers. Existence of such an autocatalytic ensemble can beverified by two dimensional gel electrophoresis and by HPLC, showing thesynthesis of a stable distribution of peptides and polypeptides. Theappropriate reaction volume depends on the number of molecular speciesused, and the concentrations necessary to favor the formation of peptidebonds over their hydrolysis. The distribution of molecular species of anautocatalytic ensemble is free to vary or change due to the emergence ofvariant autocatalytic ensembles. The peptides and polypeptides whichconstitute an autocatalytic set may have certain elements in common withthe large initial ensemble (constituted of coded peptides andpolypeptides as given by our procedure) but can also contain peptidesand polypeptides which are not coded by the ensemble of stochastic genescoding for the initial ensemble.

The set of stochastic genes whose products are necessary to establishsuch an autocatalytic set can be isolated as has been described, bysequential diminution of the library of transformed clones. In addition,an autocatalytic set can contain coded peptides initially coded by thestochastic genes and synthesized continuously in the autocatalytic set.To isolate this coded subset of peptides and proteins, the autocatalyticset can be used to obtain, through immunization in an animal, polyclonalsera recognizing a very large number of the constituents of theautocatalytic set.

These sera can be utilized to screen the library of stochastic genes tofind those genes expressing proteins able to combine with the antibodiespresent in the sera.

This set of stochastic genes expresses a large number of codedstochastic proteins which persist in the autocatalytic set. Theremainder of the coded constituents of such an autocatalytic set can beisolated by serial diminution of the library of stochastic genes, fromwhich the subset detected by immunological methods has first beensubtracted.

Such autocatalytic sets of peptides and proteins, obtained as noted, mayfind a number of practical applications.

We claim:
 1. A process for the production of an expression vector whichcomprises at least one stochastic sequence of polynucleotides,comprising the steps of:providing in an appropriate buffer at leastthree different sequences of oligonucleotides, said oligonucleotideseach comprising at least 7 nucleotide residues; polymerizing saidoligonucleotides to form a stochastic sequence of polynucleotides; andligating said stochastic sequence of polynucleotides into a linearizedexpression vector.
 2. The process according to claim 1 wherein theoligonucleotides further comprise a heptamer.
 3. The process accordingto claim 1, said process further comprising the steps of:transforming acompetent clone with said ligated expression vector; culturing saidtransformed competent clone; purifying said expression vector from saidamplified competent clone; isolating said cultured stochastic sequenceof polynucleotides from said expression vector; cutting said stochasticsequence of polynucleotides by means of at least one restriction enzymewhich corresponds to a specific restriction enzyme site present in saidstochastic sequence of polynucleotides; treating said cut stochasticsequence of polynucleotides with a T4 ligase to create a new ensemble ofstochastic sequence of polynucleotides containing a new stochasticsequence of polynucleotides; and ligating said new ensemble ofstochastic sequence of polynucleotides into an expression vector.
 4. Theprocess according to claim 2 wherein the heptamers are palindromic. 5.The process according to claim 4 wherein the palindromic heptamers areselected from the group consisting of:5' XTCGCGA 3'; 5' XCTGCAG 3'; and5' RGGTACC 3';where X=A, G, C, or T, and R=A or T.
 6. The processaccording to claim 1 wherein the oligonucleotides further comprise anoctomer.
 7. The process according to claims 1 or 6, said process furthercomprising the steps of:transforming a competent clone with said ligatedexpression vector; culturing said transformed competent clone; purifyingand isolating said expression vector from said cultured competent clone;cutting said stochastic sequence of polynucleotides by means of at leastone restriction enzyme which corresponds to said specific restrictionenzyme site present in said stochastic sequence of polynucleotides;treating said cut stochastic sequence of polynucleotides with a T4ligase to create a new ensemble of stochastic sequence ofpolynucleotides comprising a new stochastic sequence of polynucleotides;and ligating said new ensemble of stochastic sequence of polynucleotidesinto an expression vector.
 8. The process according to claim 6 whereinthe octamers are palindromic.
 9. The process according to claim 8wherein the palindromic octamers are selected from the group consistingof:5' GGAATTCC 3'; 5' GGTCGACC 3'; 5' CAAGCTTG 3'; 5' CCATATGG 3'; and5' CATCGATG 3'.
 10. The process according to claim 1 wherein thestochastic double stranded DNA fragments are about 160 to 800 base pairsin length.
 11. A process for the production of an expression vectorcapable of producing a transcription product or a translation productcomprising at least one stochastic sequence of polynucleotides,comprising the steps of:linearizing an expression vector; reacting saidlinearized expression vector with terminal transferase enzyme in thepresence of desired ratios of deoxynucleotide-triphosphates of guanine,cytosine, thymidine, and adenine to form a stochastic polynucleotidesequence at each 3' extremity of said linearized vector; hybridizingsaid stochastic polynucleotide sequence at a 3' extremity of saidlinearized vector; and synthesizing a second strand from said 3' ends ofsaid hybridized vector by incubating with polymerase.
 12. A process forthe production of a library of expression vectors capable of producing atranscription product or a translation product, said vectors comprisingat least one stochastic sequence of polynucleotides, comprising thesteps of:producing at least one stochastic sequence of polynucleotides;ligating said stochastic sequence of polynucleotides into an expressionvector; transforming a competent clone with said ligated expressionvector; culturing said transformed clone; screening and/or selectingsaid transformed clone in order to isolate a clone expressing astochastic polynucleotide leading to the synthesis of a transcriptionproduct or a translation product; isolating said selected or screenedtransformed clone; and isolating the expression vector cultured in saidselected or screened transformed clone so identified.
 13. A library ofexpression vectors capable of producing a transcription product or atranslation product, said expression vectors comprising at least onestochastic sequence of polynucleotides, produced in accordance with theprocess of claim
 12. 14. An expression vector produced in accordancewith the process of claim 1, 11,
 12. 15. The process according to claims11 or 12 wherein said translation product comprises a product having adesired property and is selected from the group consisting of a peptide,a polypeptide or a protein.
 16. The process according to claims 1 or 12wherein said transcription product comprises a product having a desiredproperty and is selected from the group consisting of a RNA or a DNA.17. The library of expression vectors according to claim 13 wherein saidlibrary comprises stochastic nucleotide sequences encoding for at least10,000 peptides, polypeptides or proteins.
 18. A method of producing adiverse population of vectors comprising:(a) synthesizing a diversepopulation of stochastically generated polynucleotide sequencescomprising greater than about 1×10⁵ different polynucleotide sequences,said method consisting of stochastic copolymerization of double strandedoligonucleotides, copolymerization of the four kinds of nucleotidetriphosphates consisting of adenine, cytosine, guaninie and thymine, andchemical synthesis, and (b) inserting said diverse population ofstochastically generated polynucleotide sequences into a population ofvectors to form a diverse population of vectors containingstochastically generated polynucleotide sequences.
 19. The method ofclaim 18, which said diverse population of stochastically generatedpolynucleotide sequences, further comprise greater than about 1×10⁶different polynucleotide sequences.
 20. The method of claim 18, whichsaid diverse population of stochastically generated polynucleotidesequences, further comprise greater than about 1×10⁷ differentpolynucleotide sequences.
 21. The method of claim 18, wherein saiddiverse population of stochastically generated polynucleotide sequences,further comprise greater than about 1×10⁸ different polynucleotidesequences.
 22. The method of claim 18, wherein said inserting furthercomprises hybridization of complementary ends.
 23. The method of claim18, wherein said inserting further comprises ligation.
 24. The method ofclaim 18, further comprising introducing said diverse population ofvectors containing stochastically generated polynucleotide sequencesinto host cells.
 25. The method of claim 18, wherein step (a) furthercomprises synthesizing a population of at least partially stochasticpolynucleotide sequences.
 26. The method of claim 18, wherein step (b)further comprises digesting the diverse population of vectors with arestriction enzyme having a recognition sequence absent in theexpression vector and reinserting the digested products into saiddigested population of vectors to form a different population having agreater number of stochastic polynucleotide sequences.
 27. A method ofproducing a diverse population of vectors, comprising stochasticallycopolymerizing a diverse population of vectors containing doublestranded polynucleotides so as to produce a new population of vectorscontaining greater than about 1×10⁵ different polynucleotide sequences.28. The method of claim 27, wherein said new population of vectorsfurther comprise greater than about 1×10⁶ different polynucleotidesequences.
 29. The method of claim 27, which said new population ofvectors further comprise greater than about 1×10⁷ differentpolynucleotide sequences.
 30. The method of claim 27, wherein said newpopulation of vectors further comprise greater than about 1×10⁸different polynucleotide sequences.
 31. The method of claim 27, whereinsaid stochastic copolymerization is effected by hybridization ofcomplementary ends.
 32. The method of claim 27, wherein said stochasticcopolymerization is effected by ligation.
 33. The method of claim 27,further comprising introducing said diverse population of vectorscontaining stochastically generated polynucleotide sequences into hostcells.
 34. The method of claim 27, wherein said double strandedpolynucleotides further comprise stochastic polynucleotide sequences.35. The method of claim 27, wherein said double stranded polynucleotidesfurther comprise at least partially stochastic polynucleotide sequences.36. The method of claim 27, wherein said diverse population of vectorsfurther comprises two or more diverse populations of vectors.
 37. Amethod of producing a diverse populations of vectors, comprising:(a)obtaining one or more diverse populations of vectors containing diversesequences of double stranded polynucleotides; (b) digesting the one ormore diverse populations of vectors with a restriction enzyme, and (c)stochastically copolymerizing the one or more diverse populations ofdouble stranded polynucleotides so as to produce a new population ofgreater than about 1×10⁵ different polynucleotide sequences.
 38. Themethod of claim 37, wherein said new population of vectors furthercomprise greater than about 1×10⁶ different polynucleotide sequences.39. The method of claim 37, wherein said new population of vectorsfurther comprise greater than about 1×10⁷ different polynucleotidesequences.
 40. The method of claim 37, wherein said new population ofvectors further comprise greater than about 1×10⁸ differentpolynucleotide sequences.
 41. The method of claim 37, wherein saidstochastic copolymerization is effected by hybridization ofcomplementary ends.
 42. The method of claim 37, wherein said stochasticcopolymerization is effected by ligation.
 43. The method of claim 37,further comprising introducing said diverse population of vectorscontaining stochastically generated polynucleotide sequences into hostcells.
 44. The method of claim 37, wherein said double strandedpolynucleotides further comprise stochastic polynucleotide sequences.45. The method of claim 37, wherein said double stranded polynucleotidesfurther comprise at least partially stochastic polynucleotide sequences.46. The method of claim 37, wherein said diverse population of vectorsfurther comprises two or more diverse populations of vectors.